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Keywords:

  • sodium arsenite;
  • yeast;
  • cell killing;
  • reactive oxygen species;
  • Ca2+

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Arsenic is a toxic metalloid that is widely distributed in the environment, and its toxicity has been demonstrated in several models. However, the mechanism of arsenic toxicity still remains unclear. In this study, the toxic effects of sodium arsenite (1–7 mM) on yeast cells were investigated. The experimental results showed that sodium arsenite inhibited yeast cell growth, and the inhibitory effect of cell growth (OD600 nm values) was positively correlated with arsenite concentrations. Sodium arsenite caused loss of cell viability in a concentration- and duration-dependent manner in yeast cells. However, arsenite-caused cell viability loss was blocked by either antioxidants (200 U mL−1 CAT and 0.5 mM AsA) or Ca2+ antagonists (0.5 mM LaCl3 and 0.5 mM EGTA). We also found intracellular reactive oxygen species (ROS) and Ca2+ levels increased significantly in yeast cells after exposure to 3 mM and 7 mM sodium arsenite for 6 h compared with the control. These results indicated that high concentrations of arsenite-induced yeast cell killing was associated with elevated levels of intracellular ROS and Ca2+.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Arsenic is a known human carcinogen that is widely distributed in the earth's crust (soil and rocks), air, water, plants, and animals in variable concentrations (Banerjee et al., 2009). Arsenic can enter human body through inhalation, ingestion, and skin contact. In general, inorganic arsenic is regarded as the most hazardous among all the arsenic species present in the environment. Inorganic arsenic exists mainly in two forms, arsenite (III) or arsenate (V), and arsenite is more toxic than arsenate (Barrett et al., 1989). Several studies have demonstrated that arsenite can induce not only chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) in animal cells (Dopp et al., 2004), plant cells (Wu et al., 2010), and human cells (Jha et al., 1992) but also cell death in cultured human and animal cells (McNeely et al., 2008; Agarwal et al., 2009).

Saccharomyces cerevisiae has been used as a useful model organism for cell death research because of a series of benefits such as not being pathogenic, rapid growth, inexpensive accessibility and simple, and well-understood genetics (Carmona-Gutierrez et al., 2010). Previous studies have shown that in yeast, cell death can also be induced not only by acetic acid (Ludovico et al., 2001), aspirin (Balzan et al., 2004), pheromones (Severin & Hyman, 2002), and hyperosmotic stress (Ribeiro et al., 2006) but also by arsenic (Du et al., 2007). Du et al. (2007) found arsenic-induced apoptosis in yeast cells is associated with increased intracellular reactive oxygen species (ROS) levels, and several other researchers have also used yeast mutants and DNA microarray to ravel the mechanisms of arsenic toxicity (Thorsen et al., 2007; Hosiner et al., 2009; Zhou et al., 2009). However, the cellular mechanism of arsenite toxicity is not clear.

Reactive oxygen species and calcium ions (Ca2+) are largely recognized as important signaling messengers that play important roles in cell death in several models such as animal cells (Brookes et al., 2004), plant cells (Yi et al., 2012), and yeast cells (Zheng et al., 2007). However, it is not clear whether arsenite-caused cytotoxicity is associated with accumulation of intracellular ROS and Ca2+, and how ROS and Ca2+ signaling involve in arsenic-caused toxicity. Therefore, yeast cells were employed to investigate the cellular mechanisms involved in arsenite-caused cytotocixity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Strains and growth conditions

Saccharomyces cerevisiae cells were grown in YPD medium containing 1% yeast extract, 2% peptone, and 2% glucose. Cell growth experiment was performed in liquid culture and incubated on a mechanical shaker (200 r.p.m.) at 30° C.

Measurement of cell growth

Cell growth was monitored by measuring the optical density of the cultures at 600 nm (OD600 nm) using a spectrophotometer. The initial OD600 nm was adjusted to 0.05 and shaken at 30° C, OD600 nm values were determined over a 24-h period. The inhibition rate (%) was calculated using the following formula: Inhibition rate (%) = (1−OD600 nm value of treated cells/OD600 nm value of untreated cells) × 100%.

Measurement of cell viability

For arsenite treatment, yeast cells were grown in fresh YPD medium containing various concentrations of sodium arsenite (NaAsO2, Merk). For other combination treatments, selected antagonists including ascorbic acid (0.5 mM AsA), catalase (200 U mL−1 CAT), Ca2+ chelator (0.5 mM ethylene glycol tetraacetic acid, EGTA), Ca2+ channel inhibitors (0.5 mM LaCl3) were, respectively, added to YPD medium in the presence of 3 mM sodium arsenite. Cell viability was measured by methylene blue staining method.

Measurement of ROS and intracellular Ca2+

The generation of intracellular ROS in yeast cells was detected using the oxidant-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) according to the methods described by Chen et al. (2003). After exposure to chemicals, yeast cells were incubated in DCFH-DA at a final concentration of 50 μM at 30° for 20 min in the dark. For determination of intracellular Ca2+ levels, a calcium fluorescent indicator fluo-3 acetomethoxyester (Fluo-3 AM) was used in this study. After treatment, yeast cells were incubated in PBS, pH 7.4, at 30° C for 50 min with 5 mM Fluo-3 AM. The levels of intracellular ROS and Ca2+ were analyzed using a fluorescence-activated cell sorter (FACS) Calibur (Becton Dickinson) at low flow rate with excitation at 488 nm. Fifty thousand cells were measured per sample.

Statistical analysis

Data were calculated as the mean of results from at least three independent experiments or one representative result of parallel experiments. Error bars represent SD. Analysis of variance (anova) and Dunnett's t test were used to determine the significant differences between the control and treatment groups.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Effect of sodium arsenite on growth of yeast cells

Effect of sodium arsenite exposure on yeast cell growth was investigated by measuring optical density at 600 nm. The results showed that a negative correlation existed between the cell density and arsenite concentration (Fig. 1). At the concentration of 1 mM, arsenite exposure have no significant effect on cell density. The cell density is decreased with increasing arsenite concentration in a range of 2–7 mM. No increase in cell density was observed in yeast cells after exposure to 7 mM arsenite, and the inhibition rate reached about 90%. These results indicated that sodium arsenite can inhibit cell division and growth.

image

Figure 1. (a) Growth curves of yeast cells in liquid YPD medium containing different concentrations of sodium arsenite. OD values were determined at 2-h intervals over 24-h period. (b) Inhibition rate was calculated as the 1 minus the relative OD600 (the OD600 value of treated cells divided by that of untreated cells).

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Sodium arsenite induces cell killing

As shown in Fig. 2, arsenite induced cell killing in yeast after exposure to 1–7 mM sodium arsenite for 3–24 h, and the cell killing rate increased with increasing arsenic concentration and exposure time. When exposed to 1 mM sodium arsenite for a short term, there was no significant cell killing, but a long-term exposure caused significant killing. However, exposure to 7 mM sodium arsenite for a short term caused significant cell killing, the killing rate reached about 80% after 24 h of exposure. These results indicated that sodium arsenite could induce cytotoxicity in yeast cells, and the toxic effect occurred basically in a time- and dose-dependent manner.

image

Figure 2. Viability assays of yeast cells exposed to arsenite. a and b indicate significant differences (a< 0.05, b< 0.01) between the control and arsenite treatment groups.

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Sodium arsenite induces intracellular ROS elevation

As shown in Fig. 3, intracellular ROS levels increased with increasing arsenite concentrations, and significant differences were observed between the control and treatment groups after exposure to 3 mM or 7 mM arsenite for 6 h. There were 15- and 37-fold increase in ROS levels above control, respectively, in 3 mM and 7 mM arsenite treatment groups. When ROS scavengers (200 U mL−1 CAT or 0.5 mM AsA) were used simultaneously with 3 mM arsenite, both ROS levels and cell killing induced by arsenite were effectively blocked (Fig. 4). These results showed a positive relationship between intracellular ROS levels and cell killing, suggesting the important role of elevated ROS in arsenite-induced yeast cell killing.

image

Figure 3. DCFH-DA fluorescence intensity, indicating the ROS levels in yeast cells measured by flow cytometric analysis after 6 h of arsentie exposure. (a) YPD control. (b) 3 mM arsenite. (c) 7 mM arsenite.

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image

Figure 4. (i) The green fluorescence of yeast cells in control samples (a and a′), in As treatment groups (b and b′) and in the combination treatments of 3 mM arsenite and 200 U mL−1 CAT (c and c′) or 0.5 mM AsA (d and d′) after DCFH-DA staining. The DCFH-DA fluorescence indicates ROS production. (ii) Changes of cell killing in yeast cells exposed to 3 mM arsenite in the presence of antioxidant (200 U mL−1 CAT or 0.5 mM AsA). a and b indicate the significant difference (a< 0.05, b< 0.01) between the control and arsenite treatment groups, c and d indicate the significant difference (c< 0.05, d< 0.01) between the arsenite treatment groups and combination treatment groups.

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Sodium arsenite induces intracellular Ca2+ elevation

To assess whether intracellular Ca2+ accumulation regulates arsenite-induced cell killing, the fluorescence intensity of Fluo-3 AM in yeast cells was investigated in three independent experiments. The results showed that the relative fluorescence intensity of intracellular Ca2+ obviously increased in yeast cells after exposure to 3 mM and 7 mM arsenite for 6 h (Fig. 5). There were 1.55- and 1.75-fold increase in Ca2+ levels above control, respectively, in 3 mM and 7 mM arsenite treatment group.

image

Figure 5. Intracellular Ca2+ fluorescence intensity was measured by flow cytometric analysis after 6 h of arsentie exposure. (a) YPD control. (b) 3 mM arsenite. (c) 7 mM arsenite.

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When yeast cells were incubated in 3 mM sodium arsenite simultaneously with 0.1 mM LaCl3 (a calcium channel blocker) or 0.5 mM EGTA (Ca2+ chelator), arsenite-induced cell killing was effectively blocked, associated with a significant decrease in Fluo-3 AM fluorescence signal of yeast cells (Fig. 6). These results indicate that sodium arsenite–induced cell killing was associated with increased intracellular Ca2+ level. A channel-mediated Ca2+ influx across the plasma membrane contributed to the elevation of intracellular Ca2+ concentration and subsequent cell killing in arsenite-treated yeast cells.

image

Figure 6. (i) The green fluorescence of yeast cells in control samples (a and a′), in As treatment group (b and b′) and in the combination treatment of 3 mM arsenite and 0.5 mM LaCl3 (c and c′) or 0.5 mM EGTA (d and d′) after Fluo-3 AM staining. The Fluo-3 AM fluorescence indicates the Ca2+ content. (ii) Changes of cell killing in yeast cells exposed to 3 mM arsenite in the presence of Ca2+ antagonists (0.5 mM LaCl3 or 0.5 mM EGTA). a and b indicate the significant difference (a< 0.05, b< 0.01) between the control and arsenite treatment groups, c and d indicate the significant difference (c< 0.05, d< 0.01) between the arsenite treatment groups and combination treatment group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Arsenic is a toxic metalloid that is widely distributed in the environment. Chronic exposure to arsenic is associated with a number of diseases, such as neurotoxicity (Vahidnia et al., 2007), birth defects (Wu et al., 2011), and metabolic disorders such as diabetes, gastrointestinal tract disorders, and cardiovascular diseases (Sarkar et al., 2003; Jana et al., 2006). However, the exact mechanism of arsenic-induced cancer and disease is not clear. Therefore, yeast cells were used to provide a better understanding of potential mechanisms underlying arsenic toxicity in the present study.

In this study, we found that high concentrations of sodium arsenite caused yeast cell viability decrease, and the relative fluorescence intensity of ROS significantly increased in yeast cells after exposure to 3 mM arsenite for 6 h, which is consistent with earlier report in Chang human hepatocytes exposed to arsenite (Li et al., 2011). When yeast cells were incubated in sodium arsenite simultaneously with CAT or AsA, arsenite induced-ROS generation and the killing rate decreased significantly vs. arsenite treatment group, indicating that intracellular ROS was involved in arsenic-induced cell killing. ROS are generated by all aerobic cells as byproducts of a number of metabolic reactions or in response to various stimuli. Excessive ROS and their byproducts can attack deoxyribose, purine and pyrimidine bases in DNA molecule resulting in DNA damage (Yi et al., 2007), which have been shown to induce cell death in yeast (Perrone et al., 2008).

Calcium ions (Ca2+) act as a universal second messenger in a variety of cells, and numerous functions of all types of cells are regulated by Ca2+. Ca2+ takes dual responsibility not only for promotion of cell death, but also for regulation of cell survival in response to a variety of pathological conditions (Shen et al., 2002; Yakimova et al., 2007). In our study, obvious increase of intracellular Ca2+ levels and the cell killing rate were observed in yeast cells after exposure to sodium arsenite. However, when either calcium channel blocker LaCl3 or Ca2+ chelator EGTA was used to block intracellular Ca2+ increase, arsenite-induced cell killing significantly decreased, which demonstrated that arsenite-caused cell killing is associated with an increased intracellular Ca2+ levels. High levels of intracellular Ca2+ can lead to enhanced generation of ROS, and elicit cell death (Zheng et al., 2007).

Arsenic exposure caused cell death in variety of cultured human cells (Han et al., 2010; Rostami et al., 2012) and animal cells (Agarwal et al., 2009). In our present study, we identified the toxic effect of sodium arsenite on yeast cells and provide the evidences of cell viability loss in arsenite-exposed yeast cells, which is similar to the effects of arsenic exposure in animal and plant cells. However, the discovery of involvement of intracellular ROS and Ca2+ in arsenite-caused cell killing might shed some new light on the mechanism of arsenic toxicity.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study was supported by the National Natural Science Foundation of China (Grant No. 30870454 and 30470318) and Shanxi Scholarship Council of China (Grant No. 2012013).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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